We recently demonstrated that cell surface action of trypsin or thrombin is sufficient to initiate cell division in nonproliferating cultures of fibroblast-like cells (1-3). Using similar procedures, I will determine whether nonproteolytic mitogens such as insulin or epidermal growth factor (EGF) can initiate cell division without direct intracellular interaction. This will involve (a) immobilizing these mitogens on polystyrene beads to prevent their internalization, (b) determining the activity of the immobilized peptides by measuring glucose uptake, thymidine incorporation and increase in cell number, (c) measuring the amount of material released from the beads into the medium using quantitation of radiolabeled peptides and assays for mitogenic activity, and (d) determining whether any material is released from beads directly into the cell using EM autoradiography. I will also continue my studies to determine how thrombin interacts with components of the cell surface to initiate cell division. Thrombin binds with high affinity to surface receptors on fibroblast-like cells and this binding appears to be necessary for initiation of cell division by thrombin (4). Therefore, I will isolate and characterize the thrombin receptor using photoaffinity techniques. Events which occur after thrombin binds to its receptor, such as receptor aggregation or capping may also play a role in initiation since several conditions alter the mitogenic response to thrombin without affecting thrombin binding. I will visualize these events using established procedures for fluorescence microscopy, EM autoradiography, and EM analysis of ferritin conjugated thrombin and evaluate whether any of these events correlate with the initiation of cell division using several independent conditions including (a) thrombin concentrations which do and do not initiate, (b) proteolytically inhibited thrombin which binds but does not initiate, (c) dexamethasone treatment which appears to enhance the mitogenic effectiveness of events subsequent to thrombin binding, and (d) cells selected using fluorescence-activated cell sorting which bind fluorescent thrombin but are not initiated.